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The effects of NO2 on the uptake and assimilation of nitrate by soybean plants
Environmental and Experimental Botany 10 (1998) 33 – 40
The effects of NO2 on the uptake and assimilation of nitrate by soybean plants Z. Qiao *, F. Murray En6ironmental Science, Murdoch Uni6ersity, Murdoch, WA 6150, Australia Received 27 August 1997; accepted 2 September 1997
Abstract Twelve-day-old soybean plants were supplied with low (1 mM) or high (5 mM) nitrate to the roots and exposed to 0 (control), 0.22 (low), or 1.1 (high) ml l − 1 NO2 for 7 days. The low NO2 treatment had no significant effects on nitrate uptake and dry weight of plants. The high NO2 treatment decreased the amount of nitrate uptake and dry weight per plant. This inhibition of nitrate uptake and growth by high NO2 was more significant for the plants growing at low nitrate than the plants growing at high nitrate. The acidity of both leaves and growth medium of the plants exposed to high NO2 was higher than that of non-exposed plants. High NO2 treatment increased the ammonium concentration in roots and decreased the amount of organic N per plant. These results suggested that exposure to high-level NO2 caused an increase in H + in the leaves of exposed plants. The H + could be partially neutralized by OH − produced from the reduction of nitrate, or exuded into the growth medium through roots. The accumulation of H + in exposed plants may inhibit nitrate uptake, ammonium assimilation, and plant growth. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Assimilation; Nitrogen dioxide; Nitrate; Soybean; Uptake
1. Introduction The effects of atmospheric NO2 on nitrate uptake by plant roots have been reported in several studies. Tomatoes and peppers exposed to NO2 showed a slight increase or no effect on root N uptake (Murray, 1984; Rowland et al., 1987). The nitrate uptake rate by barley roots was not changed by exposure to 0.3 ml l − 1 NO2 for 9 days (Rowland et al., 1987). Uptake rate of nitrate by * Corresponding author. Fax: +61 8 93104997; e-mail: [email protected]
roots of the spruce seedlings supplied with nitrate plus ammonium was decreased by 32% by exposure to 0.1 ml l − 1 NO2 for 48 h; the uptake rate by the seedlings supplied with only nitrate was just slightly depressed by the exposure (Muller et al., 1996). Exposing barley plants to 0.03 ml l − 1 NO2 for 42 days increased the amount of nitrate taken up by the plants supplied with 1 g nitrate-N per pot, but slightly decreased the nitrate uptake for the plants supplied with 3 g nitrate-N per pot (Jensen and Pilegaard, 1993). Sunflower and maize plants, exposed to 0, 0.2, 0.5 or 1.0 ml l − 1 NO2 for 2 weeks, absorbed similar amounts of
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nitrate from the soil (Okano et al., 1986). However, in the sunflower plants exposed to 2 ml l − 1 NO2 for 7 days, the amount of nitrate absorbed by roots was decreased (Okano and Totsuka, 1986). The rate-limiting step in the process of nitrate assimilation is believed to be the reduction of nitrate to nitrite (Imsade and Touraine, 1994). As NO2 exposure usually stimulates the activity of nitrate reductase (NaR) in leaves (Srivastava and Ormrod, 1984; Rowland et al., 1987; Morgan et al., 1992), stimulation of nitrate assimilation by NO2 may be expected. However, inhibition of N-assimilation and plant growth by NO2 exposure has been frequently reported (Srivastava and Ormrod, 1984; Okano et al., 1985; Hur and Wellburn, 1994). Even the stimulatory effects of very low levels of NO2 on N-assimilation can gradually disappear with prolonged exposure (Bender et al., 1991). Obviously some factor induced by NO2 other than the stimulation of the activity of NaR is acting in the process of assimilation of nitrate (Srivastava and Ormrod, 1989). This factor may be the toxic accumulation of products derived from the assimilation of atmospheric NO2 (Hur and Wellburn, 1994). A major difference between NO2 absorption by leaves and nitrate absorption by roots is that more H + are brought into plants by the former (Wellburn, 1990; Bambauer et al., 1994). The detrimental effects shown by NO2 exposure may result from H + . The aim of the present study was to test the hypothesis that the constraint on nitrate uptake in NO2-exposed plants may be concerned with the H + produced from NO2 absorption.
2. Materials and methods
2.1. Pre-culture of plants Soybean seeds (Glycine max L., cultivar ‘Oxley’) were germinated in tissue rolls. Twenty healthy seedlings were chosen for hydroponic culture. Each seedling was planted in a small plastic basket (50 mm high, 40 mm in base diameter, 58 mm in lip diameter), containing clay pellet sub-
strate to hold the seedling. Each basket was placed in one of the 20 holes (50 mm in diameter) on a hard plastic plate (420× 320× 5 mm), which served as the lid of a plastic tray (400 ×300×130 mm) containing 10 litres of nutrient solution. The roots of the seedlings were immersed in the nutrient solution which was aerated by an air pump. The solution composition was as follows (concentrations in mg l − 1): KNO3 505, KH2PO4 68, K2HPO43H2O 114, MgSO4 240, FeEDTA 33, H3BO3 2.86, MnSO4H2O 0.31, ZnSO47H2O 2.29, CuSO45H2O 0.71, CoCl2 0.22, NaMoO4 · 2H2O 0.023. The solution pH was controlled at 6.090.2 with H2SO4 or NaOH. The level of the solution in the tray was kept constant by the addition of fresh solution as required. Twelve days after germination, six healthy seedlings of similar leaf development were selected for NO2 exposure.
2.2. NO2 exposure Seven rectangular glass cuvettes (200× 200× 380 mm) and six hard PVC solution-containers (230 mm high, 120 mm in diameter) were used to hold six seedlings for fumigation. Each plant was suspended in an individual nutrient solution container. Each container, holding nutrient solution, was aerated with filtered air through a 22 gauge hypodermic needle maintained at a flow rate of 30 ml min − 1. A glass side arm with index mark and plugged with cotton wool was used to indicate solution level and a suba-seal located on the side of the container allowed sampling of the solution or topping up when required. At the beginning of exposure each container was filled with 1380 ml of nutrient solution with the same composition as the solution used for plants pre-culture except that N was supplied as 1 or 5 mM KNO3 labelled with 1–6% 15N. On the third and fifth day of fumigation about 50 ml of fresh solution was added to each container with a syringe to compensate for evaporation and uptake of the solution. Each plant was then enclosed in a rectangular glass cuvette with a detachable, split base isolating the plant top from the roots. Each cuvette contained a temperature probe and a 40 mm fan to assist mixing. An inlet, connected to the fumigant
Z. Qiao, F. Murray / En6ironmental and Experimental Botany 39 (1998) 33–40
gas supply system, and outlet were located at diagonally opposite corners of the cuvette. The plant was sealed to the base and the base sealed to the cuvette with tape to prevent gaseous exchange between the plant tops and the nutrient solution. The entire assembly was placed in a constant temperature room (30 91°C) and illuminated by a timer-controlled 1000 W metal halide lamp directed through a water filter designed to minimise temperature effects. Each cuvette inlet was connected to a flowmeter delivering charcoalfiltered air at 1 l min − 1. The flow meters, used to deliver nitrogen dioxide, were connected to a mixing chamber into which NO2 was introduced by a Brooks Instrument Div. mass flow control valve model 5850 TR. The NO2 in the mixing chamber was supplied from a NO2 cylinder. The cuvette outlet was connected to a two way solenoid valve allowing the gas to be either vented to the atmosphere or directed to a Monitor Labs 8840 nitrogen oxides analyser with a flow rate of 500 ml min − 1. An identical but empty cuvette was used to monitor adsorption of fumigation gases onto the surfaces of the cuvette. Extra solenoid valves allowed the inlet gases to be diverted to the analyser. All plumbing materials used in the study were of glass, Teflon or 316 stainless steel. The system for sampling NO2 in cuvettes was controlled by an electronic timer which switched individual valves in sequence every 10 min throughout the exposure period, enabling the analyser to measure the concentration of NO2 from cuvettes. Data were logged on a Unidata model 7000B macro data logger which recorded cuvette number, temperature, PAR (photosynthetic active radiation), concentrations of CO2, water vapour, and nitrogen oxides. Each batch of plants for exposure included three fumigated (exposed to NO2) and three control (exposed to filtered air), supplied with the same concentrations of nitrate to roots (1 mM or 5 mM). For each concentration of nitrate, three or four batches of plants were used for exposure. For each batch of plants the fumigation lasted for 7 days, 12 h per day with the photoperiod
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synchronous with the fumigation from 06:00 to 18:00. The average PAR was 320 mmol m − 2 s − 1 during daytime. The NO2 concentration at the inlets of cuvettes was demonstrated to be equal to the concentration at the outlet of the empty cuvette. The average concentration of NO2 in the fumigated chambers was maintained at two levels (ml l − 1): 0.22 (low) and 1.1 (high).
2.3. Har6est and measurements During harvest each plant was separated into leaves, stem and roots. The leaves were rinsed with deionised water, then blotted with tissues. Each leaf was cut into halves, one of which was weighed, cut into small pieces, ground in a mortar with pestle, and mixed with deionised water weighing 20 times that of the leaves. The pH of the slurry was measured with Model 407A Ionalyzer (Orion Research) and J5992-42 combination pH electrode (Cole-Parmer). The accuracy and repeatability is estimated to be 0.02 pH unit. The pH of nutrient solutions was also measured at harvest. The roots, stem, and other half-leaf were dried in a forced draught oven for 65 h at 70°C. The dry plant parts were weighed, then ground into a fine powder with a vibrating mill for determination of nitrate, nitrite, and organic nitrogen (Singh, 1988; Srivastava et al., 1994). The ammonium in the plant powders was extracted with the hot water method (Roberts et al., 1980) and determined with the phonate spectrometric method (Clesceri et al., 1989). The 15N atom% abundance and concentration of total-N in the plant powders were determined with a mass-spectrometer, the ‘Europa Scientific’ automatic nitrogen and carbon analyser system (Jensen, 1991).
2.4. Calculation and analysis The content of the N derived from atmospheric NO2 (NO2-N) was calculated with the mass balance method (Rogers et al., 1979; Hanson and Lindberg, 1991). The content of the N derived from root nitrate (nitrate-N) and uptake rate of nitrate was calculated by the 15N dilution method (Jensen and Pilegaard, 1993):
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Table 1 Nitrate uptake by roots of Glycine max, growing in a nutrient solution with 1 or 5 mM KNO3, and fumigated at three NO2 levels NO2 (ml l−1)
0 0.22 1.1
Amount of nitrate uptake (mg N per plant)
Uptake rate on root dry weight basis (mg N g−1 dry weight day−1)
1 mM nitrate
5 mM nitrate
1 mM nitrate
5 mM nitrate
16.7a 16.2a 10.8b
29.1a 28.0a 22.0b
14.7a 15.0a 11.7a
22.4a 21.7a 19.4a
Means of a parameter not followed by the same letter are significantly different at level pB0.05.
Amount of nitrate-N in a plant = total-N ×
atom% 15Nexcess in plant atom% 15Nexcess in 15N-labelled nitrate
Two-way ANOVA and the Duncan’s multiple range test were used to test the significance of the differences among different treatments (Beyer, 1974; Ott, 1977). Each value in the data tables represents the mean of three or four replicated experiments with each treatment. Each experiment contained three individual plants of the same treatment. 3. Results
3.1. Uptake of nitrate by roots The amount of nitrate taken up per plant and the uptake rates were not significantly affected by exposure to low NO2. However, the amount of nitrate uptake per plant was decreased by exposure to high NO2; the relative decrement of nitrate uptake was more significant for the plants grown at 1 mM nitrate than that for the plants at 5 mM nitrate (Table 1).
Low NO2 had no significant effect on the ammonium concentration in plants. High NO2 resulted in increased ammonium concentration in roots of plants, and in the leaves of plants grown at low nitrate (Table 2).
3.3. Content of organic N Low NO2 had no effect on the organic N content of the plants, or the concentration of organic N in leaves and roots, with the exception that it slightly increased the concentration of organic N in the leaves of plants grown at low nitrate. High NO2 decreased the total amount of organic N per plant for plants grown at both low and high nitrate, and the concentration of organic N in the roots of plants grown at high nitrate. However the concentration of organic N in leaves was increased by high NO2 (Table 3).
3.4. Dry weight of plants High NO2 resulted in decreased dry weight of plants. The most decrement in dry weight caused by high NO2 occurred in the roots of plants grown at low nitrate. Low NO2 had no statistically significant effects on dry weight of plants (Table 4).
3.2. Concentrations of inorganic N in plants 3.5. pH of lea6es and growth medium Compared to the non-fumigated plants, low NO2 had no significant effects on the nitrate concentration in plants. High NO2 increased the nitrate concentration in the plants grown at low nitrate, in contrast to the plants grown at high nitrate (Table 2).
Low NO2 had no significant effects on the acidities of leaves and nutrient solution (Table 5). High NO2 decreased leaf pH at both levels of nitrate. The pH of nutrient solution of the plants exposed to high NO2 was much lower than that of
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Table 2 Concentrations of nitrate and ammonium (mg N g−1 dry weight) in plants of Glycine max, growing in a nutrient solution with 1 or 5 mM KNO3, and fumigated at three NO2 levels NO2 (ml l−1)
Roots
Leaves 1 mM nitrate
0 0.22 1.1
5 mM nitrate
1 mM nitrate
5 mM nitrate
NH4-N
NO3-N
NH4-N
NO3-N
NH4-N
NO3-N
NO3-N
NH4-N
2.12a 2.50ab 3.02b
0.12a 0.15ab 0.20b
5.59a 5.34a 5.66a
0.23a 0.20a 0.26a
6.13a 7.28ab 8.05b
0.22a 0.27a 0.41b
11.3a 10.2a 9.7a
0.38a 0.44ab 0.50b
Means of a parameter not followed by the same letter are significantly different at level pB0.05.
the control plants. The pH of the nutrient solution of the plants not exposed to high NO2 rose during the fumigation period from an initial pH of about 5.62 to higher than 6.5, but the pH of the nutrient solution of the plants exposed to high NO2 declined slightly during the fumigation (Table 5).
4. Discussion Previous studies have reported that exposure to 5 ml l − 1 NO2 acidified the leaf sap of pea plants (Zeevaart, 1976). Similarly, our 1.1 ml l − 1 NO2 treatment increased the acidity of exposed leaves (Table 5). This increase in H + in leaves may result from the formation of HNO3 and HNO2 from NO2 dissolved in extracellular fluid (Wellburn, 1990; Bambauer et al., 1994). The pH of nutrient solution for the plants exposed to high NO2 was much lower than that for the unexposed (control) plants (Table 5). A reason for this difference in pH of nutrient solution was the decrease in nitrate uptake by roots of the plants exposed to high NO2 (Table 1), because nitrate uptake by roots is known to decrease acidity of growth medium (Lewis, 1986; Touraine et al., 1994). The plants exposed to high NO2 took up a considerable amount of nitrate during the exposure, although this uptake of nitrate, compared with the control plants, was decreased by exposure to high NO2 (Table 1). Therefore, the nutrient solution of the plants exposed to high NO2
may be expected to be obviously alkalised by the nitrate uptake. However, the pH of the nutrient solution of the plants exposed to high NO2 showed a small decline rather than an increase (Table 5). This suggested that the nutrient solution of the plants exposed to high NO2 obtained some H + during the NO2 exposure. Since the nutrient solution could not contact directly with NO2, the H + may originate from the dissolution of high NO2 in extracellular fluid of leaves and exude to nutrient solution through roots. An increase in acidity of nutrient solution is favourable for nitrate uptake by roots (Vessey et al., 1990). So the decline in pH of the nutrient solution could not be responsible for the decrease in nitrate uptake by roots of the plants exposed to high NO2 (Tables 1 and 5). The inhibition of nitrate uptake by high NO2 treatment may be a result of the increase in H + concentration in the exposed plants. As ammonium assimilation to amino acids produces H + (Raven and Smith, 1976; Raven, 1988), the increase in acidity in exposed plants may inhibit ammonium assimilation, and result in ammonium accumulation (Table 2; Yu et al., 1988). The ammonium accumulation can increase efflux of nitrate out of roots, which has the effect of decreasing net uptake of nitrate (Rajasekhar and Oelmuller, 1987; Chaillou et al., 1994). A way of nitrate uptake by plant roots is exchange with HCO3− (Touraine et al., 1990, 1992, 1994). The increase in H + concentration in the plants exposed to high NO2 may decrease the influx of nitrate by converting the HCO3− in roots
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Table 3 Content of organic N in plants of Glycine max, growing in a nutrient solution with 1 or 5 mM KNO3, and fumigated at three NO2 levels NO2 (ml l−1)
Concentration of organic N (mg N g−1 dry weight)
Amount of organic N (mg N per plant)
Leaves
0 0.22 1.1
Roots
1 mM NO3
5 mM NO3
1 mM NO3
5 mM NO3
1 mM NO3
5 mM NO3
44.6a 45.4a 41.5b
53.2a 53.8a 50.1b
52.4a 55.0b 55.6b
56.8a 56.2a 59.5b
31.7a 32.5a 33.0a
38.3a 36.9ab 35.6b
Means of a parameter not followed by the same letter are significantly different at level pB0.05.
to H2CO3. According to the dissociation constant of carbonic acid (Ka1 =4.30 ×10 − 7), the concentration of HCO3− is 1.6 times the concentration of H2CO3 when the pH is 6.57 (about the pH of leaves of the control plants; Table 5); if pH declines to 6.37 (about the pH of leaves of the plants exposed to high NO2; Table 5), the HCO3− concentration is equal to the H2CO3 concentration. The increase in H + concentration in exposed plants may also inhibit the synthesis of organic acids (including malate) in shoot, which would further decrease the HCO3− in roots, since the HCO3− is formed by decarboxylation of the malate from shoot (Touraine et al., 1990, 1992, 1994). In the low NO2 treatment, the nitrate produced from NO2 absorption is rapidly reduced in leaves (Srivastava and Ormrod, 1989; Segschneider et al., 1995). However, exposure to high NO2 caused increases in nitrate concentration and acidity in the exposed leaves especially for the plants grown
at low nitrate supply (Tables 2 and 5). This suggested that the assimilation capacity was exceeded at the high NO2 concentration. The accumulation of H + in the plants exposed to high NO2 (Table 5), formed by absorption of large amount of NO2, may result in ammonium accumulation (Table 2; Yu et al., 1988), which can bring about a loss of NaR activity (Orebamjo and Stewart, 1975; Padidam et al., 1991), and cause an accumulation of nitrate in the plants at low nitrate supply (Table 2). The OH − produced by reduction of the nitrate taken up by roots can offset the H + produced by NO2 assimilation, so the plants grown at high nitrate showed less decline in nitrate reduction power than the plants grown at low nitrate (Table 2). The high NO2 treatment caused ammonium accumulation (Table 2) and a decrease in amount of organic N in exposed plants (Table 3). This appears to result from the inhibition of ammonium assimilation by increased leaf acidity associ-
Table 4 Dry weight (mg per plant) of Glycine max, growing in a nutrient solution with 1 or 5 mM KNO3, and fumigated at three NO2 levels NO2 (ml l−1)
0 0.22 1.1
1 mM nitrate
5 mM nitrate
Total
Leaves
Roots
Total
Leaves
Roots
740a 726a 653b
366a 360a 321b
188a 165a 92b
815a 782a 716b
401a 387a 349b
130a 129a 113b
Means of a parameter not followed by the same letter are significantly different at level pB0.05.
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Table 5 pH of leaves and nutrient solution for Glycine max, growing in a nutrient solution with 1 or 5 mM KNO3, and fumigated at three NO2 levels NO2 (ml l−1)
0 0.22 1.1
pH of leaves
pH of growth solution
1 mM nitrate
5 mM nitrate
1 mM nitrate
5 mM nitrate
6.52a 6.52a 6.34b
6.60a 6.60a 6.48b
6.54a 6.54a 5.46b
6.90a 6.88a 5.54b
The initial pH of the nutrient solution was about 5.62. Means of a parameter not followed by the same letter are significantly different at level pB0.05.
ated with exposure to high NO2. Compared with the control plants, the amount of organic N decreased more for the plants grown at low nitrate than those grown at high nitrate (Table 3), since reduction of the nitrate from roots can produce OH − to buffer the impact of the H + from NO2 absorption. To summarize, NO2 absorption by leaves forms nitrate, nitrite and H + in plants. Much of the H + produced from NO2 absorption is consumed by the assimilation of the nitrate and nitrite from NO2. The excess H + may be removed by two processes: (1) The assimilation of the nitrate absorbed by roots produces OH − which can neutralise the excess H + . (2) The excess H + is exuded through roots into the growth medium. If the excess H + cannot be promptly removed, its accumulation would inhibit nitrate uptake by roots, retard ammonium assimilation to organic N, and slow down plant growth.
Acknowledgements Kelvin Maybury is gratefully acknowledged for technical assistance. We are grateful to Kevin Bronson for his kind help in determination of isotope ratio with mass spectrometry.
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Okano, K., Totsuka, T., Fukuzawa, T., Tazaki, T., 1985. Growth response of plants to various concentrations of nitrogen dioxide. Environ. Pollut. 38, 361–373. Okano, K., Fukuzawa, T., Tazaki, T., Totsuka, T., 1986. 15N dilution method for estimating the absorption of atmospheric NO2 by plants. New Phytol. 102, 73–84. Orebamjo, T.O., Stewart, G.R., 1975. Ammonium inactivation of nitrate reductase in Lemna minor L. Planta 122, 37 – 44. Ott, L., 1977. An Introduction to Statistical Methods and Data Analysis. Duxbury Press, North Scituate, MA. Padidam, M., Venkateswarlu, K., Johri, M.M., 1991. Ammonium represses NADPH nitrate reductase in the moss Funaria hygrometrica. Plant Sci. 75, 185–194. Rajasekhar, V.K., Oelmuller, R., 1987. Regulation of induction of nitrate reductase and nitrite reductase in higher plants. Physiol. Plant 71, 517–521. Raven, J.A., 1988. Acquisition of nitrogen by the shoots of land plants: its occurrence and implications for acid – base regulation. New Phytol. 109, 1–20. Raven, J.A., Smith, F.A., 1976. Nitrogen assimilation and transport in vascular land plants in relation to intracellular pH regulation. New Phytol. 76, 415–431. Roberts, H.M., Reisenauer, H.M., Carlson, R.M., 1980. Determination of ammonium, amides, and soluble carboxylates in plant tissues. J. Plant Nutr. 2, 395–405. Rogers, H.H., Jeffries, H.E., Witherspoon, A.M., 1979. Measuring air pollutant uptake by plants: nitrogen dioxide. J. Environ. Qual. 8, 551–557. Rowland, A.J., Drew, M.C., Wellburn, A.R., 1987. Foliar entry and incorporation of atmospheric nitrogen dioxide into barley plants of different nitrogen status. New Phytol. 107, 357 – 371. Segschneider, H., Wildt, J., Forstel, H., 1995. Uptake of 15 NO2 by sunflower (Helianthus annuus) during exposures in light and darkness: quantities, relationship to stomatal aperture and incorporation into different nitrogen pools within the plant. New Phytol. 131, 109–119.
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Singh, J.P., 1988. A rapid method for determine of nitrate in soil and plant extracts. Plant Soil 110, 137 – 139. Srivastava, H.S., Ormrod, D.P., 1984. Effects of nitrogen dioxide and nitrate nutrition on growth and nitrate assimilation in bean leaves. Plant Physiol. 76, 418 – 423. Srivastava, H.S., Ormrod, D.P., 1989. Nitrogen dioxide and nitrate nutrition effects on nitrate reductase activity and nitrate content of bean leaves. Environ. Exp. Bot. 29, 433 – 438. Srivastava, H.S., Ormrod, O.P., Hale, B.A., 1994. Responses of greening bean seedling leaves to nitrogen dioxide and nutrient nitrate supply. Environ. Pollut. 86, 37 – 42. Touraine, B., Grignon, N., Grignon, C., 1990. Interaction between nitrate assimilation in shoots and nitrate uptake by roots of soybean plants: role of carboxylate. Plant Soil 124, 169 – 174. Touraine, B., Muller, B., Grignon, C., 1992. Effect of phloemtranslocated malate on NO3− uptake by roots of intact soybean plants. Plant Physiol. 99, 1118 – 1123. Touraine, B., Clarkson, D.T., Muller, B., 1994. Regulation of nitrate uptake at the whole plant level. In: Roy, J., Garnier, E. (Eds.), A Whole Plant Perspective on Carbon–Nitrogen Interactions. SPB Academic Publishing, The Hague, pp. 11 – 30. Vessey, J.K., Henry, L.T., Chaillou, S., Rager, C.D. Jr., 1990. Root-zone acidity affects relative uptake of nitrate and ammonium from mixed nitrogen sources. J. Plant Nutr. 13, 95 – 116. Wellburn, A.R., 1990. Why are atmospheric oxides of nitrogen usually phytotoxic and not alternative fertilisers? New Phytol. 115, 395 – 429. Yu, S., Li, L., Shimazaki, K., 1988. Response of spinach and kidney bean plants to nitrogen dioxide. Environ. Pollut. 55, 1 – 13. Zeevaart, A.J., 1976. Some effects of fumigating plants for short periods with NO2. Environ. Pollut. 11, 97 – 108.
The effects of NO2 on the uptake and assimilation of nitrate by soybean plants Z. Qiao *, F. Murray En6ironmental Science, Murdoch Uni6ersity, Murdoch, WA 6150, Australia Received 27 August 1997; accepted 2 September 1997
Abstract Twelve-day-old soybean plants were supplied with low (1 mM) or high (5 mM) nitrate to the roots and exposed to 0 (control), 0.22 (low), or 1.1 (high) ml l − 1 NO2 for 7 days. The low NO2 treatment had no significant effects on nitrate uptake and dry weight of plants. The high NO2 treatment decreased the amount of nitrate uptake and dry weight per plant. This inhibition of nitrate uptake and growth by high NO2 was more significant for the plants growing at low nitrate than the plants growing at high nitrate. The acidity of both leaves and growth medium of the plants exposed to high NO2 was higher than that of non-exposed plants. High NO2 treatment increased the ammonium concentration in roots and decreased the amount of organic N per plant. These results suggested that exposure to high-level NO2 caused an increase in H + in the leaves of exposed plants. The H + could be partially neutralized by OH − produced from the reduction of nitrate, or exuded into the growth medium through roots. The accumulation of H + in exposed plants may inhibit nitrate uptake, ammonium assimilation, and plant growth. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Assimilation; Nitrogen dioxide; Nitrate; Soybean; Uptake
1. Introduction The effects of atmospheric NO2 on nitrate uptake by plant roots have been reported in several studies. Tomatoes and peppers exposed to NO2 showed a slight increase or no effect on root N uptake (Murray, 1984; Rowland et al., 1987). The nitrate uptake rate by barley roots was not changed by exposure to 0.3 ml l − 1 NO2 for 9 days (Rowland et al., 1987). Uptake rate of nitrate by * Corresponding author. Fax: +61 8 93104997; e-mail: [email protected]
roots of the spruce seedlings supplied with nitrate plus ammonium was decreased by 32% by exposure to 0.1 ml l − 1 NO2 for 48 h; the uptake rate by the seedlings supplied with only nitrate was just slightly depressed by the exposure (Muller et al., 1996). Exposing barley plants to 0.03 ml l − 1 NO2 for 42 days increased the amount of nitrate taken up by the plants supplied with 1 g nitrate-N per pot, but slightly decreased the nitrate uptake for the plants supplied with 3 g nitrate-N per pot (Jensen and Pilegaard, 1993). Sunflower and maize plants, exposed to 0, 0.2, 0.5 or 1.0 ml l − 1 NO2 for 2 weeks, absorbed similar amounts of
S0098-8472/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 9 8 - 8 4 7 2 ( 9 7 ) 0 0 0 2 3 - 3
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Z. Qiao, F. Murray / En6ironmental and Experimental Botany 39 (1998) 33–40
nitrate from the soil (Okano et al., 1986). However, in the sunflower plants exposed to 2 ml l − 1 NO2 for 7 days, the amount of nitrate absorbed by roots was decreased (Okano and Totsuka, 1986). The rate-limiting step in the process of nitrate assimilation is believed to be the reduction of nitrate to nitrite (Imsade and Touraine, 1994). As NO2 exposure usually stimulates the activity of nitrate reductase (NaR) in leaves (Srivastava and Ormrod, 1984; Rowland et al., 1987; Morgan et al., 1992), stimulation of nitrate assimilation by NO2 may be expected. However, inhibition of N-assimilation and plant growth by NO2 exposure has been frequently reported (Srivastava and Ormrod, 1984; Okano et al., 1985; Hur and Wellburn, 1994). Even the stimulatory effects of very low levels of NO2 on N-assimilation can gradually disappear with prolonged exposure (Bender et al., 1991). Obviously some factor induced by NO2 other than the stimulation of the activity of NaR is acting in the process of assimilation of nitrate (Srivastava and Ormrod, 1989). This factor may be the toxic accumulation of products derived from the assimilation of atmospheric NO2 (Hur and Wellburn, 1994). A major difference between NO2 absorption by leaves and nitrate absorption by roots is that more H + are brought into plants by the former (Wellburn, 1990; Bambauer et al., 1994). The detrimental effects shown by NO2 exposure may result from H + . The aim of the present study was to test the hypothesis that the constraint on nitrate uptake in NO2-exposed plants may be concerned with the H + produced from NO2 absorption.
2. Materials and methods
2.1. Pre-culture of plants Soybean seeds (Glycine max L., cultivar ‘Oxley’) were germinated in tissue rolls. Twenty healthy seedlings were chosen for hydroponic culture. Each seedling was planted in a small plastic basket (50 mm high, 40 mm in base diameter, 58 mm in lip diameter), containing clay pellet sub-
strate to hold the seedling. Each basket was placed in one of the 20 holes (50 mm in diameter) on a hard plastic plate (420× 320× 5 mm), which served as the lid of a plastic tray (400 ×300×130 mm) containing 10 litres of nutrient solution. The roots of the seedlings were immersed in the nutrient solution which was aerated by an air pump. The solution composition was as follows (concentrations in mg l − 1): KNO3 505, KH2PO4 68, K2HPO43H2O 114, MgSO4 240, FeEDTA 33, H3BO3 2.86, MnSO4H2O 0.31, ZnSO47H2O 2.29, CuSO45H2O 0.71, CoCl2 0.22, NaMoO4 · 2H2O 0.023. The solution pH was controlled at 6.090.2 with H2SO4 or NaOH. The level of the solution in the tray was kept constant by the addition of fresh solution as required. Twelve days after germination, six healthy seedlings of similar leaf development were selected for NO2 exposure.
2.2. NO2 exposure Seven rectangular glass cuvettes (200× 200× 380 mm) and six hard PVC solution-containers (230 mm high, 120 mm in diameter) were used to hold six seedlings for fumigation. Each plant was suspended in an individual nutrient solution container. Each container, holding nutrient solution, was aerated with filtered air through a 22 gauge hypodermic needle maintained at a flow rate of 30 ml min − 1. A glass side arm with index mark and plugged with cotton wool was used to indicate solution level and a suba-seal located on the side of the container allowed sampling of the solution or topping up when required. At the beginning of exposure each container was filled with 1380 ml of nutrient solution with the same composition as the solution used for plants pre-culture except that N was supplied as 1 or 5 mM KNO3 labelled with 1–6% 15N. On the third and fifth day of fumigation about 50 ml of fresh solution was added to each container with a syringe to compensate for evaporation and uptake of the solution. Each plant was then enclosed in a rectangular glass cuvette with a detachable, split base isolating the plant top from the roots. Each cuvette contained a temperature probe and a 40 mm fan to assist mixing. An inlet, connected to the fumigant
Z. Qiao, F. Murray / En6ironmental and Experimental Botany 39 (1998) 33–40
gas supply system, and outlet were located at diagonally opposite corners of the cuvette. The plant was sealed to the base and the base sealed to the cuvette with tape to prevent gaseous exchange between the plant tops and the nutrient solution. The entire assembly was placed in a constant temperature room (30 91°C) and illuminated by a timer-controlled 1000 W metal halide lamp directed through a water filter designed to minimise temperature effects. Each cuvette inlet was connected to a flowmeter delivering charcoalfiltered air at 1 l min − 1. The flow meters, used to deliver nitrogen dioxide, were connected to a mixing chamber into which NO2 was introduced by a Brooks Instrument Div. mass flow control valve model 5850 TR. The NO2 in the mixing chamber was supplied from a NO2 cylinder. The cuvette outlet was connected to a two way solenoid valve allowing the gas to be either vented to the atmosphere or directed to a Monitor Labs 8840 nitrogen oxides analyser with a flow rate of 500 ml min − 1. An identical but empty cuvette was used to monitor adsorption of fumigation gases onto the surfaces of the cuvette. Extra solenoid valves allowed the inlet gases to be diverted to the analyser. All plumbing materials used in the study were of glass, Teflon or 316 stainless steel. The system for sampling NO2 in cuvettes was controlled by an electronic timer which switched individual valves in sequence every 10 min throughout the exposure period, enabling the analyser to measure the concentration of NO2 from cuvettes. Data were logged on a Unidata model 7000B macro data logger which recorded cuvette number, temperature, PAR (photosynthetic active radiation), concentrations of CO2, water vapour, and nitrogen oxides. Each batch of plants for exposure included three fumigated (exposed to NO2) and three control (exposed to filtered air), supplied with the same concentrations of nitrate to roots (1 mM or 5 mM). For each concentration of nitrate, three or four batches of plants were used for exposure. For each batch of plants the fumigation lasted for 7 days, 12 h per day with the photoperiod
35
synchronous with the fumigation from 06:00 to 18:00. The average PAR was 320 mmol m − 2 s − 1 during daytime. The NO2 concentration at the inlets of cuvettes was demonstrated to be equal to the concentration at the outlet of the empty cuvette. The average concentration of NO2 in the fumigated chambers was maintained at two levels (ml l − 1): 0.22 (low) and 1.1 (high).
2.3. Har6est and measurements During harvest each plant was separated into leaves, stem and roots. The leaves were rinsed with deionised water, then blotted with tissues. Each leaf was cut into halves, one of which was weighed, cut into small pieces, ground in a mortar with pestle, and mixed with deionised water weighing 20 times that of the leaves. The pH of the slurry was measured with Model 407A Ionalyzer (Orion Research) and J5992-42 combination pH electrode (Cole-Parmer). The accuracy and repeatability is estimated to be 0.02 pH unit. The pH of nutrient solutions was also measured at harvest. The roots, stem, and other half-leaf were dried in a forced draught oven for 65 h at 70°C. The dry plant parts were weighed, then ground into a fine powder with a vibrating mill for determination of nitrate, nitrite, and organic nitrogen (Singh, 1988; Srivastava et al., 1994). The ammonium in the plant powders was extracted with the hot water method (Roberts et al., 1980) and determined with the phonate spectrometric method (Clesceri et al., 1989). The 15N atom% abundance and concentration of total-N in the plant powders were determined with a mass-spectrometer, the ‘Europa Scientific’ automatic nitrogen and carbon analyser system (Jensen, 1991).
2.4. Calculation and analysis The content of the N derived from atmospheric NO2 (NO2-N) was calculated with the mass balance method (Rogers et al., 1979; Hanson and Lindberg, 1991). The content of the N derived from root nitrate (nitrate-N) and uptake rate of nitrate was calculated by the 15N dilution method (Jensen and Pilegaard, 1993):
Z. Qiao, F. Murray / En6ironmental and Experimental Botany 39 (1998) 33–40
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Table 1 Nitrate uptake by roots of Glycine max, growing in a nutrient solution with 1 or 5 mM KNO3, and fumigated at three NO2 levels NO2 (ml l−1)
0 0.22 1.1
Amount of nitrate uptake (mg N per plant)
Uptake rate on root dry weight basis (mg N g−1 dry weight day−1)
1 mM nitrate
5 mM nitrate
1 mM nitrate
5 mM nitrate
16.7a 16.2a 10.8b
29.1a 28.0a 22.0b
14.7a 15.0a 11.7a
22.4a 21.7a 19.4a
Means of a parameter not followed by the same letter are significantly different at level pB0.05.
Amount of nitrate-N in a plant = total-N ×
atom% 15Nexcess in plant atom% 15Nexcess in 15N-labelled nitrate
Two-way ANOVA and the Duncan’s multiple range test were used to test the significance of the differences among different treatments (Beyer, 1974; Ott, 1977). Each value in the data tables represents the mean of three or four replicated experiments with each treatment. Each experiment contained three individual plants of the same treatment. 3. Results
3.1. Uptake of nitrate by roots The amount of nitrate taken up per plant and the uptake rates were not significantly affected by exposure to low NO2. However, the amount of nitrate uptake per plant was decreased by exposure to high NO2; the relative decrement of nitrate uptake was more significant for the plants grown at 1 mM nitrate than that for the plants at 5 mM nitrate (Table 1).
Low NO2 had no significant effect on the ammonium concentration in plants. High NO2 resulted in increased ammonium concentration in roots of plants, and in the leaves of plants grown at low nitrate (Table 2).
3.3. Content of organic N Low NO2 had no effect on the organic N content of the plants, or the concentration of organic N in leaves and roots, with the exception that it slightly increased the concentration of organic N in the leaves of plants grown at low nitrate. High NO2 decreased the total amount of organic N per plant for plants grown at both low and high nitrate, and the concentration of organic N in the roots of plants grown at high nitrate. However the concentration of organic N in leaves was increased by high NO2 (Table 3).
3.4. Dry weight of plants High NO2 resulted in decreased dry weight of plants. The most decrement in dry weight caused by high NO2 occurred in the roots of plants grown at low nitrate. Low NO2 had no statistically significant effects on dry weight of plants (Table 4).
3.2. Concentrations of inorganic N in plants 3.5. pH of lea6es and growth medium Compared to the non-fumigated plants, low NO2 had no significant effects on the nitrate concentration in plants. High NO2 increased the nitrate concentration in the plants grown at low nitrate, in contrast to the plants grown at high nitrate (Table 2).
Low NO2 had no significant effects on the acidities of leaves and nutrient solution (Table 5). High NO2 decreased leaf pH at both levels of nitrate. The pH of nutrient solution of the plants exposed to high NO2 was much lower than that of
Z. Qiao, F. Murray / En6ironmental and Experimental Botany 39 (1998) 33–40
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Table 2 Concentrations of nitrate and ammonium (mg N g−1 dry weight) in plants of Glycine max, growing in a nutrient solution with 1 or 5 mM KNO3, and fumigated at three NO2 levels NO2 (ml l−1)
Roots
Leaves 1 mM nitrate
0 0.22 1.1
5 mM nitrate
1 mM nitrate
5 mM nitrate
NH4-N
NO3-N
NH4-N
NO3-N
NH4-N
NO3-N
NO3-N
NH4-N
2.12a 2.50ab 3.02b
0.12a 0.15ab 0.20b
5.59a 5.34a 5.66a
0.23a 0.20a 0.26a
6.13a 7.28ab 8.05b
0.22a 0.27a 0.41b
11.3a 10.2a 9.7a
0.38a 0.44ab 0.50b
Means of a parameter not followed by the same letter are significantly different at level pB0.05.
the control plants. The pH of the nutrient solution of the plants not exposed to high NO2 rose during the fumigation period from an initial pH of about 5.62 to higher than 6.5, but the pH of the nutrient solution of the plants exposed to high NO2 declined slightly during the fumigation (Table 5).
4. Discussion Previous studies have reported that exposure to 5 ml l − 1 NO2 acidified the leaf sap of pea plants (Zeevaart, 1976). Similarly, our 1.1 ml l − 1 NO2 treatment increased the acidity of exposed leaves (Table 5). This increase in H + in leaves may result from the formation of HNO3 and HNO2 from NO2 dissolved in extracellular fluid (Wellburn, 1990; Bambauer et al., 1994). The pH of nutrient solution for the plants exposed to high NO2 was much lower than that for the unexposed (control) plants (Table 5). A reason for this difference in pH of nutrient solution was the decrease in nitrate uptake by roots of the plants exposed to high NO2 (Table 1), because nitrate uptake by roots is known to decrease acidity of growth medium (Lewis, 1986; Touraine et al., 1994). The plants exposed to high NO2 took up a considerable amount of nitrate during the exposure, although this uptake of nitrate, compared with the control plants, was decreased by exposure to high NO2 (Table 1). Therefore, the nutrient solution of the plants exposed to high NO2
may be expected to be obviously alkalised by the nitrate uptake. However, the pH of the nutrient solution of the plants exposed to high NO2 showed a small decline rather than an increase (Table 5). This suggested that the nutrient solution of the plants exposed to high NO2 obtained some H + during the NO2 exposure. Since the nutrient solution could not contact directly with NO2, the H + may originate from the dissolution of high NO2 in extracellular fluid of leaves and exude to nutrient solution through roots. An increase in acidity of nutrient solution is favourable for nitrate uptake by roots (Vessey et al., 1990). So the decline in pH of the nutrient solution could not be responsible for the decrease in nitrate uptake by roots of the plants exposed to high NO2 (Tables 1 and 5). The inhibition of nitrate uptake by high NO2 treatment may be a result of the increase in H + concentration in the exposed plants. As ammonium assimilation to amino acids produces H + (Raven and Smith, 1976; Raven, 1988), the increase in acidity in exposed plants may inhibit ammonium assimilation, and result in ammonium accumulation (Table 2; Yu et al., 1988). The ammonium accumulation can increase efflux of nitrate out of roots, which has the effect of decreasing net uptake of nitrate (Rajasekhar and Oelmuller, 1987; Chaillou et al., 1994). A way of nitrate uptake by plant roots is exchange with HCO3− (Touraine et al., 1990, 1992, 1994). The increase in H + concentration in the plants exposed to high NO2 may decrease the influx of nitrate by converting the HCO3− in roots
Z. Qiao, F. Murray / En6ironmental and Experimental Botany 39 (1998) 33–40
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Table 3 Content of organic N in plants of Glycine max, growing in a nutrient solution with 1 or 5 mM KNO3, and fumigated at three NO2 levels NO2 (ml l−1)
Concentration of organic N (mg N g−1 dry weight)
Amount of organic N (mg N per plant)
Leaves
0 0.22 1.1
Roots
1 mM NO3
5 mM NO3
1 mM NO3
5 mM NO3
1 mM NO3
5 mM NO3
44.6a 45.4a 41.5b
53.2a 53.8a 50.1b
52.4a 55.0b 55.6b
56.8a 56.2a 59.5b
31.7a 32.5a 33.0a
38.3a 36.9ab 35.6b
Means of a parameter not followed by the same letter are significantly different at level pB0.05.
to H2CO3. According to the dissociation constant of carbonic acid (Ka1 =4.30 ×10 − 7), the concentration of HCO3− is 1.6 times the concentration of H2CO3 when the pH is 6.57 (about the pH of leaves of the control plants; Table 5); if pH declines to 6.37 (about the pH of leaves of the plants exposed to high NO2; Table 5), the HCO3− concentration is equal to the H2CO3 concentration. The increase in H + concentration in exposed plants may also inhibit the synthesis of organic acids (including malate) in shoot, which would further decrease the HCO3− in roots, since the HCO3− is formed by decarboxylation of the malate from shoot (Touraine et al., 1990, 1992, 1994). In the low NO2 treatment, the nitrate produced from NO2 absorption is rapidly reduced in leaves (Srivastava and Ormrod, 1989; Segschneider et al., 1995). However, exposure to high NO2 caused increases in nitrate concentration and acidity in the exposed leaves especially for the plants grown
at low nitrate supply (Tables 2 and 5). This suggested that the assimilation capacity was exceeded at the high NO2 concentration. The accumulation of H + in the plants exposed to high NO2 (Table 5), formed by absorption of large amount of NO2, may result in ammonium accumulation (Table 2; Yu et al., 1988), which can bring about a loss of NaR activity (Orebamjo and Stewart, 1975; Padidam et al., 1991), and cause an accumulation of nitrate in the plants at low nitrate supply (Table 2). The OH − produced by reduction of the nitrate taken up by roots can offset the H + produced by NO2 assimilation, so the plants grown at high nitrate showed less decline in nitrate reduction power than the plants grown at low nitrate (Table 2). The high NO2 treatment caused ammonium accumulation (Table 2) and a decrease in amount of organic N in exposed plants (Table 3). This appears to result from the inhibition of ammonium assimilation by increased leaf acidity associ-
Table 4 Dry weight (mg per plant) of Glycine max, growing in a nutrient solution with 1 or 5 mM KNO3, and fumigated at three NO2 levels NO2 (ml l−1)
0 0.22 1.1
1 mM nitrate
5 mM nitrate
Total
Leaves
Roots
Total
Leaves
Roots
740a 726a 653b
366a 360a 321b
188a 165a 92b
815a 782a 716b
401a 387a 349b
130a 129a 113b
Means of a parameter not followed by the same letter are significantly different at level pB0.05.
Z. Qiao, F. Murray / En6ironmental and Experimental Botany 39 (1998) 33–40
39
Table 5 pH of leaves and nutrient solution for Glycine max, growing in a nutrient solution with 1 or 5 mM KNO3, and fumigated at three NO2 levels NO2 (ml l−1)
0 0.22 1.1
pH of leaves
pH of growth solution
1 mM nitrate
5 mM nitrate
1 mM nitrate
5 mM nitrate
6.52a 6.52a 6.34b
6.60a 6.60a 6.48b
6.54a 6.54a 5.46b
6.90a 6.88a 5.54b
The initial pH of the nutrient solution was about 5.62. Means of a parameter not followed by the same letter are significantly different at level pB0.05.
ated with exposure to high NO2. Compared with the control plants, the amount of organic N decreased more for the plants grown at low nitrate than those grown at high nitrate (Table 3), since reduction of the nitrate from roots can produce OH − to buffer the impact of the H + from NO2 absorption. To summarize, NO2 absorption by leaves forms nitrate, nitrite and H + in plants. Much of the H + produced from NO2 absorption is consumed by the assimilation of the nitrate and nitrite from NO2. The excess H + may be removed by two processes: (1) The assimilation of the nitrate absorbed by roots produces OH − which can neutralise the excess H + . (2) The excess H + is exuded through roots into the growth medium. If the excess H + cannot be promptly removed, its accumulation would inhibit nitrate uptake by roots, retard ammonium assimilation to organic N, and slow down plant growth.
Acknowledgements Kelvin Maybury is gratefully acknowledged for technical assistance. We are grateful to Kevin Bronson for his kind help in determination of isotope ratio with mass spectrometry.
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